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IN VITRO CULTURE of POINTED GOURD (Trichosanthes Dioica Roxb.)

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IN VITRO CULTURE of POINTED GOURD (Trichosanthes Dioica Roxb.) ISSN 0258-7122 Bangladesh J. Agril. Res. 35(1) : 135-142, March 2010 IN VITRO CULTURE OF POINTED GOURD (Trichosanthes dioica Roxb.) M. A. MALEK1, D. KHANAM2, M. KHATUN3 M. H. MOLLA4 AND M. A. MANNAN4 Abstract An experiment was conducted to study the in vitro culture of pointed gourd. Cotyledon rescued from physiologically matured seeds (PMS) and immatured seeds (IMS) of pointed gourd were used as explants. Cotyledon excised from PMS responded very well in all culture conditions. Plant regenerated from cotyledon of PMS ranged from 38 to 96% in different hormonal formulations of culture media. Highest percentage of shoot regeneration was observed in MS + 1.0 mg/l BAP and lowest in MS + 2.5 mg/l BAP. No plant regeneration was observed in cotyledon from immatured seeds. The highest percentage of root induction (99%) was achieved in half MS medium supplemented with 0.5 mg/l NAA. The regenerated plantlets were successfully established in earthen pot. Keywords: Cotyledon, in vitro, pointed gourd. Introduction Pointed gourd (Trichosanthes dioica Roxb.) is an under exploited important summer vegetables in Bangladesh (Rashid, 1993). It is one of the most nutritive cucurbit vegetables that holds a coveted position in the vegetable market during summer and rainy season (Singh et al., 1992). Being very rich in protein and Vitamin A, it has certain medicinal properties and many reports are available regarding its role in circulatory system, especially in lowering blood sugar and serum triglycerides (Sheshadri, 1990). In recent years, in vitro procedures are used to some degree in almost every major agronomic, vegetables, and fiber crops (Hoque et al., 1998). The success of this technology requires an efficient protocol for plant regeneration from isolated organs, tissues, and cells (Islam et al., 1994). The success of a crop improvement programme, depends on selection of desirable plants, which is possible if wide variation is present in the base population. But there is less variability in pointed gourd (Uddin, 2000), Variability can be created by somaclonal variation or by in vitro polyploidization (Hoque et al., 1998). So, it is necessary to develop in vitro plant regeneration protocol for pointed gourd. But less attention has been given to tissue culture of pointed gourd than its closely related taxa, such as cucumber and melon (Dong and Jia, 1991). Plantlet formation has already been reported in Cucurbita pepo 1Plant Genetic Resources Centre, BARI, Joydebpur, Gazipur 1701, 2, 3 & 4Biotechnology Division, BARI, Joydebpur, Gazipur 1701, 5Farm Division, BARI, Joydebpur, Gazipur 1701, Bangladesh. 136 MALEK et al. (Jelaska, 1974), water melon (Dong and Jia, 1991), cucumber (Chee, 1990; Gambley and Dodd, 1990) and Momordica charantia (Islam et al., 1994). Therefore, the present investigation was conducted with a view to developing a protocol for plant regeneration through in vitro culture of pointed gourd. Materials and Method The study was conducted in the Tissue Culture Laboratory, Biotechnology Division, BARI, Joydebpur, Gazipur 1701. The experiments were conducted following Completely Randomized Design (CRD). The data were subjected to mean value plus Standard Error (S. E). Plant materials Cotyledon rescued from matured (PMS) and immatured seeds (IMS) of pointed gourd were considered as explants for this study. The explants were collected from the experimental field of pointed gourd, Plant Genetic Resources Centre (PGRC), BARI, Joydebpur, Gazipur 1701. Preparation of explant Fourteen to seventeen days old fruits of pointed gourd having physiologically matured seeds (PMS) with solid cotyledons were collected from the experimental field of pointed gourd, PGRC, BAR!, Gazipur. The fruits were cut and the seeds removed from the fruits with the help of sterilized scalpel. The seed coats were removed carefully with the help of sterilized forceps and knife and the cotyledons were taken in a beaker. Eight to twelve days old fruits having IMS with soft seed coat and semi solid cotyledons were collected from the same experimental field and prepared according to the previous methods. Surface sterilization of explant Prepared cotyledons were surface sterilized gently with distilled water for 3-4 times. The surface sterilized cotyledons were dipped into 70% ethyl alcohol for 30 seconds. Finally, the cotyledons were washed 3 times with sterilized distilled water inside the clean bench and these cotyledons (excluding embryo) were placed in the culture medium. Inoculation The excised solid and semi-solid cotyledons (excluding embryo) were inoculated in each culture test tubes containing MS media with various concentrations of BAP (0.5, 1.0, 1.5. 2.0, and 2.5 mg/l) and NAA (0.5, 1.0, 1.5. 2.0, and 2.5 mg/l) for in vitro shoot regeneration. The pH of the medium was adjusted to 5.7 ± 0.1 using 0.1 N sodium hydroxide (NaOH) or 0.1 N HCI. In order to solidify the media, laboratory grade agar of 8.0 g (0.8%) was added to the solution. The IN VITRO CULTURE OF POINTED GOURD 137 culture tubes were plugged with aluminium foil and marked with glass marker pen to indicate specific hormonal supplement. The culture tubes were sterilized at 1.09 kg/cm2 pressure at 121°C for 15 minutes in an autoclave. After auto claving, the culture media were taken out and allowed to cool and solidify. For growth and development of cultures, the temperature was set 25±1°C at a light intensity of 2000-3000 lux from fluorescent tubular lamps and the photoperiod was maintained generally 16 hours light and 8 hours dark (16 L/8 D) and relative humidity 60-70%. Successful shoot formation become evident when small green fresh leaves began to emerge. Subcultures carried out regularly at an interval of 4-5 weeks. The shoot induction percentages, days to shoot initiation, shoot number per explant, and shoot length have been recorded after 4 weeks of culture. In vitro 1 grown shoots of pointed gourd were cultured in /2 MS medium supplemented with different concentration of NAA (0.1, 0.2, 0.3, 0.4, and 0.5 mg/l) for root initiation. The well rooted plantlets were then kept in room temperature for 2-3 days and transferred to polyethylene bags containing a mixture of soil, sand, and rice bran ash (1:1:1) and moist them adequately for proper hardening. Results and Discussion The choice of explants is of cardinal importance and makes an absolute difference between success and failure in inducing regeneration in vitro. In most of the cucurbits, the root induction was achieved on either basal MS medium alone or with very low level of auxin (Mythili and Thomas, 1999). In the present experiment, different concentrations of BAP and NAA were used singly to investigate the induction of shoot and its subsequent regeneration. Data on shoot induction percentage, days to shoot initiation, shoot number per explant and shoot length per explant were recorded after 4 weeks of culture. The results are presented under the following separate heads: Shoot induction and proliferation The response of cotyledon explants from matured and immatured seeds of pointed gourd cultured in MS media supplemented with BAP (0.5, 1.0, 1.5, 2.0, and 2.5 mg/l) and NAA (0.5, 1.0, 1.5, 2.0, and 2.5 mg/l) are shown in Table 1. The cotyledon obtained from matured seeds when used as the explant produced roots and shoots. The highest percentage of shoot induction (96.00%) and highest number of shoot per explant (2.55) were obtained in MS medium supplemented with 1.0 mg/l BAP followed by 1.0 mg/l NAA (82.00%, 2.30) and the lowest percentage of shoot induction (40.00%) recorded in 2.5 mg/l BAP. The higher shoot formation was observed in MS + 1.0 mg/l BAP. The results proved that BAP has a positive effect on shoot induction. Shoot development occurred within 14 days in 1.0 mg/l BAP, whereas it took 17 days in 1.0 mg/l NAA. Similar 138 MALEK et al. results were reported by Hoque et al. (1998) in pointed gourd. They also observed roots and shoots in hormone free MS medium which is not similar to the results observed in this experiment. Table 1. Effect of different concentrations of BAP and NAA on shoot induction and proliferation from cotyledon of matured and immatured seeds of pointed gourd. Shoot Days to Shoot Treatment Shoot length Explant induction shoot number per (mg/l) (cm) (%) initiation explant Cotyledon Control 0 0 0 0 from BAP 0.5 65.00 14 2.20* 3.72 ** matured 1.0 96.00 ** 14 2.55 ** 3.81** seeds 1.5 55.00 14 2.00 3.10 2.0 41.00 14 1.50 2.00 2.5 40.00 14 1.00 1.45 Mean 59.40 14.00 1.85 2.82 S.E(±) 9.176 0.000 0243 0.420 Cotyledon Control 0 0 0 0 from NAA 0.5 68.00 17 2.10 3.31 ** matured 1.0 82.00 ** 17 2.30 ** 3.57 ** seeds 1.5 56.00 17 2.27 2.56 2.0 47.00 17 2.00 2.20 2.5 43.00 17 1.48 2.10 Mean 59.20 17.00 2.03 2.74 S.E(±) 6.383 0.000 0.132 0.264 Cotyledon Control 0 0 0 0 from BAP 0.5 0 0 0 0 immatured 1.0 0 0 0 0 seeds 1.5 0 0 0 0 2.0 0 0 0 0 2.5 0 0 0 0 Cotyledon Control 0 0 0 0 from NAA 0.5 0 0 0 0 immatured 1.0 0 0 0 0 seeds 1.5 0 0 0 0 2.0 0 0 0 0 2.5 0 0 0 0 *, ** Significant at 5% and 1% level, respectively.
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